This protocol is a variant of the Hanahan protocol <cite>Hanahan91</cite> using CCMB80 buffer for DH10B and TOP10 strains. It builds on Example 2 of the [[Media:pat6855494.pdf | Bloom05 patent]] as well.

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{{back to protocols}}

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==Overview==

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This protocol is a variant of the Hanahan protocol <cite>Hanahan91</cite> using CCMB80 buffer for DH10B, TOP10 and MachI strains. It builds on Example 2 of the [[Media:pat6855494.pdf | Bloom05 patent]] as well. This protocol has been tested on TOP10, MachI and [[Talk:TOP10 chemically competent cells|BL21(DE3)]] cells. See [[Bacterial Transformation]] for a more general discussion of other techniques. The [[Media:pat6960464.pdf | Jesse '464 patent]] describes using this buffer for DH5&alpha; cells. The [[Media:pat6709852.pdf | Bloom04]] patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells.

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'''This is the chemical transformation protocol used by [[User:Tk|Tom Knight]] and the [http://partsregistry.org Registry of Standard Biological Parts].

Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic

Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic

must be detergent free for these protocols. The easiest way to do this is to avoid washing

must be detergent free for these protocols. The easiest way to do this is to avoid washing

glassware, and simply rinse it out. Autoclaving glassware filled 3/4 with DI water is an effective

glassware, and simply rinse it out. Autoclaving glassware filled 3/4 with DI water is an effective

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way to remove most detergent residue. Media and buffers should be prepared in detergent free glassware

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way to remove most detergent residue. Media and buffers should be prepared in detergent free glassware and cultures grown up in detergent free glassware.

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and cultures grown up in detergent free glassware.

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====Prechill plasticware and glassware====

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Prechill 250mL centrifuge tubes and screw cap tubes before use.

===Preparing seed stocks===

===Preparing seed stocks===

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* streak TOP10 cells on an [[SOB]] plate and grow for single colonies at 23 C

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* Streak TOP10 cells on an [[SOB]] plate and grow for single colonies at 23&deg;C

** room temperature works well

** room temperature works well

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* Pick single colonies into 2 ml of SOB medium and shake overnight at 23 C

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* Pick single colonies into 2 ml of SOB medium and shake overnight at 23&deg;C

** room temperature works well

** room temperature works well

* Add glycerol to 15%

* Add glycerol to 15%

Line 17:

Line 41:

* Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes

* Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes

** This step may not be necessary

** This step may not be necessary

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* Place in -80 freezer indefinitely.

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* Place in -80&deg;C freezer indefinitely.

===Preparing competent cells===

===Preparing competent cells===

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* Inoculate 250 ml of [[SOB]] medium with 1 ml vial of seed stock and grow at 20 C to an OD of 0.5

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* Inoculate 250 ml of [[SOB]] medium with 1 ml vial of seed stock and grow at 37&deg;C to an OD600nm of 0.5

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** This takes approximately 16 hours. Controlling the temperature makes this a more reproducible process, but is not essential. Room temperature will work. You can adjust this temperature somewhat to fit your schedule

** Previous protocol indicated amount of acetic acid added should be 23 ml/liter but that amount was found to be 2X too much per tests on 1.23.07 --[[User:Meaganl|Meaganl]] 15:50, 25 January 2007 (EST)

Overview

This protocol is a variant of the Hanahan protocol [1] using CCMB80 buffer for DH10B, TOP10 and MachI strains. It builds on Example 2 of the Bloom05 patent as well. This protocol has been tested on TOP10, MachI and BL21(DE3) cells. See Bacterial Transformation for a more general discussion of other techniques. The Jesse '464 patent describes using this buffer for DH5α cells. The Bloom04 patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells.

Procedure

Preparing glassware and media

Eliminating detergent

Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic
must be detergent free for these protocols. The easiest way to do this is to avoid washing
glassware, and simply rinse it out. Autoclaving glassware filled 3/4 with DI water is an effective
way to remove most detergent residue. Media and buffers should be prepared in detergent free glassware and cultures grown up in detergent free glassware.

Prechill plasticware and glassware

Prechill 250mL centrifuge tubes and screw cap tubes before use.

Preparing seed stocks

Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C

room temperature works well

Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C

room temperature works well

Add glycerol to 15%

Aliquot 1 ml samples to Nunc cryotubes

Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes

This step may not be necessary

Place in -80°C freezer indefinitely.

Preparing competent cells

Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 37°C to an OD600nm of 0.5

This takes approximately 16 hours.

Controlling the temperature makes this a more reproducible process, but is not essential.

Room temperature will work. You can adjust this temperature somewhat to fit your schedule

Aim for lower, not higher OD if you can't hit this mark

Centrifuge at 5000rpm at 4°C for 10 minutes in a flat bottom centrifuge bottle.

Flat bottom centrifuge tubes make the fragile cells much easier to resuspend

It is often easier to resuspend pellets by mixing before adding large amounts of buffer

Discard supernatant by pouring out slowly and pipeting remains

Gently resuspend in 80 ml of ice cold CCMB80 buffer

sometimes this is less than completely gentle. It still works.

Incubate on ice 20 minutes

Centrifuge again at 4°C and discard supernatant as described above.

Resuspend in 10 ml of ice cold CCMB80 buffer.

Test OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells.